Method and apparatus for multi-parameter data analysis

ABSTRACT

In one aspect, the present invention relates to a method  200  for identifying one or more phenotypes from a multi-parameter data set. The method  200  comprises measuring  202  correlation between pairs of parameters within the multi-parameter data set, modifying  204  correlated parameter values within a predetermined multi-parameter data analysis set to form an analysis parameter set, and analyzing  206  the multi-parameter data set using the analysis parameter set to identify one or more phenotypes from the multi-parameter data set. Various embodiments of the present invention may, for example, be used in an automated high-content screening (HCS) apparatus  100  for biological cellular analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/EP2010/055439 filed Apr.23, 2010, published on Oct. 28, 2010 as WO 2010/122147, which claimspriority to application number 0907079.8 filed in Great Britain on Apr.24, 2009.

FIELD OF THE INVENTION

The present invention relates generally to a method and apparatus foranalysis of multi-parameter data. More specifically, the presentinvention relates to the analysis of multi-parameter data in order toidentify one or more phenotypes. Using such phenotypes, variousembodiments of the present invention may, for example, be used inautomated high-content screening (HCS) that uses biological cellularimage analysis.

BACKGROUND OF THE INVENTION

HCS, the application of automated sub-cellular imaging and imageanalysis to investigating cellular signalling pathways and processes (S.A. Haney, P. LaPan, J. Pan and J. Zhang, “High-content screening movesto the front of the line,” Drug Discovery Today, Vol. 11, No. 19-20, pp.889-894, October 2006), is becoming widely adopted across both industryand academia as a rapid and cost-effective route to generating highlyinformative biological data. HCS provides investigators with powerfultechnologies and applications for detailed investigation of cellularbiology in-situ and in-context, and as a consequence generates largemulti-parameter data sets corresponding, for example, to variousrespective images.

In many studies the full potential of this data has not been fullyexplored or exploited. Standard methods of data analysis and comparison,such as the use of mean and standard deviation, which have beenroutinely used in high-throughput screening (HTS), obscure underlyingpatterns and trends in HCS data by averaging cellular populationresponses.

A simple example of this obscuration occurs in chemical inhibitor orRNAi studies, where, for example, a 50% decrease measured by HTS metricsas a mean response may represent 50% inhibition in all cells or,alternatively, 100% inhibition in 50% of cells, with the remainder beingunaffected.

The situation is further worsened by the typical distributions ofcellular intensity or spatial data, which is rarely, if ever, normallydistributed, thus making mean and standard deviation a poor descriptorof the data distribution.

Consequently comparison of HCS data between samples based on averagedresponses is not only underutilising the data but is also likely to beinaccurate in many cases.

Limitations of standard data averaging techniques have led to theadoption of various non-parametric analysis methods, such as use of theKolmogorov-Smirnov (KS) distance (S. Siegel and N. J. Castellan,Non-Parametric Statistics for the Behavioural Sciences, McGraw-Hill, NewYork, USA, 2^(nd) Edition, 1988) for comparing cell population data anddistributions in HCS data (Z. E. Perlman, M. D. Slack, Y. Feng, T. J.Mitchison, L. F. Wu and S. J. Altschuler, “Multidimensional drugprofiling by automated microscopy,” Science, Vol. 306, pp. 1194-1198, 12Nov. 2004; and B. Zhang, X. Gu, U. Uppalapati, M. A. Ashwell, D. S.Leggett and C. J. Li, “High-content fluorescent-based assay forscreening activators of DNA damage checkpoint pathways,” Journal ofBiomolecular Screening, Vol. 13, No. 6, pp. 538-543, 19 Jun. 2008).

For example, US 2006/0154236 (Altschuler et al) describe methods andsystems for the analysis of cells based on the automated collection ofdata from image processing software and statistical analysis of thisdata. The methods described include the use of intra-sample KS distanceas a measure of population differences and means for normalising KSdistance by dividing by a measure of the variability of the descriptor(e.g. standard deviation) within a population.

However, whilst the use of such non-parametric data analysis methods isan improvement on previous techniques, there still remains the need forboth faster and more accurate data analysis techniques, particularly foranalysing the extremely large multi-parameter data sets typicallygenerated by HCS/HTS.

SUMMARY OF THE INVENTION

The present invention has thus been devised whilst bearing theabove-mentioned drawbacks associated with conventional data analysismethods in mind.

According to a first aspect of the present invention, there is provideda method for identifying one or more phenotypes from a multi-parameterdata set. The method comprises measuring correlation between pairs ofparameters within the multi-parameter data set, modifying correlatedparameter values within a predetermined multi-parameter data analysisset to form an analysis parameter set, and analysing the multi-parameterdata set using the analysis parameter set to identify one or morephenotypes from the multi-parameter data set.

According to a second aspect of the present invention, there is providedan apparatus for automated high-content screening (HCS) of one or moremulti-parameter data sets. The apparatus comprises a processor that isoperable to measure correlation between pairs of parameters within amulti-parameter data set. The processor is also operable to modifycorrelated parameter values within a predetermined multi-parameter dataanalysis set for the multi-parameter data set to form an analysisparameter set and to analyse the multi-parameter data set using theanalysis parameter set to identify one or more phenotypes from themulti-parameter data set.

Modification of correlated parameter values within a predeterminedmulti-parameter data analysis set may comprise multiplication or otherarithmetic modification of the parameter values using one or morefactors derived from correlation measurements, including modification ofthe parameter value to zero, or removal of the parameter from furtheranalysis, for example.

By determining phenotypes from an analysis parameter set that is formedof independent, or substantially non-correlated, parameters overall dataprocessing time is reduced.

Moreover, this technique additionally provides for more accuratemeasurement of phenotypes thereby enabling improved feature recognitionto be provided automatically at high speed, for example, in a HCS/HTSsystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an apparatus for automated high-content screening (HCS) ofone or more multi-parameter data sets in accordance with an embodimentof the present invention.

FIG. 2 shows a method for identifying one or more phenotypes from amulti-parameter data set in accordance with various aspects of thepresent invention.

FIG. 3 illustrates the principle of determining the Kolmogorov-Smimov(KS) distance between two data populations in accordance with variousembodiments of the present invention.

FIG. 4 shows an illustration of intra-sample and inter-sample KSdistance determinations as used in various embodiments of the presentinvention.

FIG. 5 shows the relationship between data parameters and phenotype mapdimensions in an embodiment of the present invention.

FIG. 6 shows representative images from high-content screening of cellstreated with increasing concentrations of Mitomycin C used to derivedata for analysis in accordance with an aspect of the present invention.

FIG. 7 shows nuclear area data derived from high-content screening ofcells treated with increasing concentrations of Mitomycin C used toderive data for analysis in accordance with an aspect of the presentinvention.

FIG. 8 shows nuclear perimeter data derived from high-content screeningof cells treated with increasing concentrations of Mitomycin C used toderive data for analysis in accordance with an aspect of the presentinvention.

FIG. 9 shows nuclear weighted relative moment of inertia data derivedfrom high-content screening of cells treated with increasingconcentrations of Mitomycin C used to derive data for analysis inaccordance with an aspect of the present invention.

FIG. 10 shows nuclear length data derived from high-content screening ofcells treated with increasing concentrations of Mitomycin C used toderive data for analysis in accordance with an aspect of the presentinvention.

FIG. 11 shows intra-sample KS distance measures data derived fromhigh-content screening of cells treated with increasing concentrationsof Mitomycin C in accordance with an aspect of the present invention.

FIG. 12 shows inter-sample KS distance measures data derived fromhigh-content screening of cells treated with increasing concentrationsof Mitomycin C in accordance with an aspect of the present invention.

FIG. 13 shows phenotype maps derived from intra-sample and inter-sampleKS distance measurements in accordance with an aspect of the presentinvention.

FIG. 14 shows breakdown of phenotype maps into sub-components inaccordance with an aspect of the present invention.

FIG. 15 shows phenotype scores derived from phenotype maps for cellstreated with increasing concentrations of MMC in accordance with anaspect of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows an apparatus 100 for automated high-content screening (HCS)of one or more multi-parameter data sets in accordance with anembodiment of the present invention. The apparatus 100, which isillustrated schematically for clarity, comprises a light source 102 forproducing light 120 a.

The light 120 a is focussed by a condenser 104 onto a test plate 108.The test plate 108 may contain an array of wells or spots 109 to beimaged. The condenser 104 can focus the light 120 b in a focal plane atthe test plate 108. The test plate 108 may be provided as a consumableproduct, and the spots 109 might contain various materials that are ableto interact with certain types of cells (e.g. mammalian cells).

In various embodiments, the test plate 108 may comprise at least onefiducial marker (not shown) provided to aid in aligning the test plate108 within the apparatus 100. For example, one or more coloured dyes maybe provided within the spots 109. Such coloured dyes can be identifiedby various imaging systems in order to derive data relating to therelative positioning of the test plate 108 within the apparatus 100. Forexample, the apparatus 100 may be a GE INCELL ANALYZER 1000™ that iscommercially available from GE Healthcare Life Sciences, LittleChalfont, Buckinghamshire, U.K., and which can use four colour channelsto image the test plate 108. One colour channel may thus be dedicated toimaging coloured fiducial markers provided in various of the spots 109in order to obtain data relating to the positioning of the test plate108 within the apparatus 100.

The apparatus 100 also contains a detector system 112 and a translationmechanism (not shown). The translation mechanism is configured to movethe focus of the light 120 b relative to the test plate 108 (e.g. bymoving the test plate 108 in the x-y plane). This enables a plurality ofimages to be acquired from respective of the individual spots 109.Additionally, the translation mechanism may also be operable to move thetest plate 108 in the z-direction shown in FIG. 1, for example, in orderto bring the spots 109 into focus.

For certain embodiments, only one spot is imaged at a time. The imagesacquired are of sufficient magnification to resolve cells andsub-cellular morphology. With the current GE INCELL ANALYZER 1000™, thismay entail use of a 20× objective, the field of view of which isslightly smaller than a single spot. However, various methods of theinvention would also work for lower power magnification imaging, e.g. onGE INCELL ANALYZER 1000™ using a 4× objective to image 4-6 spots/image.

An aperture stop 106 is optionally provided between the light source 102and the detector system 112, the size of which may be variable. Forexample, various differently sized movable apertures may be rotated intoposition or a continuously variable iris-type diaphragm may be provided.Image contrast can be controlled by changing the aperture setting of theaperture stop 106.

Focussed light 120 b passing through the aperture stop 106 passesthrough the sample test plate 108 in a transmission imaging mode.Emergent light 120 c modulated with image information relating tomaterial adjacent to an individual spot 109 is collected by an objectivelens 110 and focussed 120 d onto the detector system 112, and is used toform an original image for that spot 109.

Various embodiments of methods of the present invention are independentof the imaging modality used, e.g. they can operate with transmission orreflection geometry. For GE INCELL ANALYZER 1000™ imaging anepi-fluorescence mode may be used, with both the fiducial marker spotsand the assay signals from the cells being imaged at differentexcitation and emission wavelengths. However there is nothing inprinciple to prevent a mix of imaging modes being deployed, providedthat they do not interfere. For example, it would be possible to use anon-fluorescent dye for fiducial marking and to detect the fiducialmarks by absorbance in reflectance or transmission geometry, whiledetecting assay signals by epi-fluorescence.

The detector system 112 is operable to acquire a plurality of imagesfrom the test plate 108, each image may be represented in electronicform as a respective multi-parameter data set. For example, severalmulti-parameter data sets may be obtained each representing an image ofrespective different spots 109 or of the same spot 109 at differentpoints in time. Differences between neighbouring spots 109 or temporalchanges occurring within the same spot 109 can thus be analysed.

The detector system 112 is also operably coupled to a processor 114 thatin turn is operable to process the multi-parameter data sets. Theprocessor 114 is operable to measure correlation between pairs ofparameters within a multi-parameter data set. For example, parameterssuch as cell perimeter, diameter, ellipticity, etc. may be assessed in apair-wise fashion to determine a quantified degree of correlationtherebetween.

The processor 114 is then operable to modify correlated parameter valueswithin a predetermined multi-parameter data analysis set for themulti-parameter data set to form an analysis parameter set. Thepredetermined multi-parameter data analysis set may, for example,contain a list of all parameters that can be measured for a particularcell image. Such a list may then, for example, be pruned by theprocessor 114 to remove one of a pair of parameters having a correlationthreshold greater than a predetermined value. For example, the processor114 may determine that a cell feature's perimeter and diameter have acorrelation greater than 95% and thus remove perimeter as a parameter tobe determined from the multi-parameter data set. An appropriatelyrevised list of parameters to be determined is stored as the analysisparameter set.

The processor 114 is also operable to analyse the multi-parameter dataset using the analysis parameter set to identify one or more phenotypesfrom the multi-parameter data set. For example, the processor 114 maysequentially determine a value for each parameter in the analysisparameter set for each appropriate cell feature. Where such a cellfeature occurs many times in an image defined by a multi-parameter dataset, any pruning of the original predetermined multi-parameter dataanalysis set ensures that data processing overheads are reduced.Analysis of the multi-parameter data set is thereby quickened.Additionally, identification of various features is also statisticallyimproved by the removal of any correlated parameters from the finalmulti-parameter data set analysis. Moreover, such a technique alsoprovides dynamic/automatic modification of the parameters used for dataanalysis such that they are not necessarily predetermined but caninstead adapt to the data that is being analysed.

The processor 114 may be further operable to identify one or morephenotypes from a plurality of multi-parameter data sets and comparerespective phenotypes from the multi-parameter data sets to identifyvariations therebetween. For example, the processor 114 may be used tocompare phenotypes from one image to those of another image. Such atechnique enables control samples of cells provided at the spots 109 tobe compared to similar but treated cells provided at others of the spots109.

An advantageous option for the apparatus 100 is to provide a processor114 that is further operable to form one or more respective analysisparameter sets for a plurality of multi-parameter data sets, and tocompare the analysis parameter sets to determine whether or not acorrelation relationship is maintained between the multi-parameter datasets. By determining whether spatial (e.g. across an array of spots 109)and/or temporal variations in the correlation between various parametersoccurs, extra information that is not conventionally available can beobtained.

For example, a cell nucleus may die (e.g. via apoptosis) and split intosmall fragments. These may generally have the same overall shape (e.g.round/ovoid/etc.). In this case, the parameters relating to imageddiameter and perimeter will have a relatively high degree of correlationfor both the initial cell nucleus and the fragments. However, during theapoptosis process, the nucleus may change shape into a stellate(star-like) form, and in this latter form a measurement of correlationbetween the parameters relating to diameter and perimeter will drop.Hence by monitoring the level of correlation between various phenotypes,additional potentially useful information relating to various biologicalprocesses can be obtained.

In various embodiments, correlation may be determined by using anon-parametric statistical pair-wise measurement made between thephenotypes in the predetermined multi-parameter data analysis set. Forexample, a Kolmogorov-Smirnov (KS) distance measurement analysis, as isdescribed in further detail below, may be used. Use of the KS distancemeasurement analysis technique is preferred in certain embodiments as itis relatively fast. However, those skilled in the art will be aware thatvarious other correlation measurement techniques could instead be used.

Additionally, the processor 114 can be configured to control atranslation mechanism (not shown) to move the focal position of thelight source 102 relative to the spot plate 108. The processor 114 may,for example, be provided as part of a conventional computer systemappropriately programmed to perform such tasks, or may be provided by adigital signal processor (DSP), dedicated application specificintegrated circuit (ASIC), appropriately configured firmware, etc.

The apparatus 100 of various embodiments of the present invention maycomprise a microscope with one or more cameras and a processor 114 thatcan be used as a high-content screening apparatus. Images represented byone or more multi-parameter data sets may be generated by the apparatus100, or may provided from storage, transmitted etc. to the processor114, for automatic analysis. Various ways for providing such automatedanalysis are described in greater detail below by way of non-limitingexample.

FIG. 2 shows a method 200 for identifying one or more phenotypes from amulti-parameter data set in accordance with various aspects of thepresent invention. The multi-parameter data set may include datacorresponding to an image provided, for example, using an apparatus 100of the type described in connection with FIG. 1, above.

The method 200 includes the step 202 of measuring correlation betweenpairs of parameters within the multi-parameter data set. Then at step204 correlated parameters from within a predetermined multi-parameterdata analysis set are modified to form an analysis parameter set.

Modification of correlated parameter values may comprise multiplicationor other arithmetic modification of the parameter values using one ormore factors derived from correlation measurements, includingmodification of the parameter value to zero, or removal of the parametercompletely from further analysis, for example.

At step 206 the multi-parameter data set is analysed using the analysisparameter set to identify one or more phenotypes from themulti-parameter data set. Various techniques for providing thefunctionality of method 200 are described in greater detail below.

The method 200 may be used to determine whether changes occur betweenmulti-parameter data analysis sets. For example, the method 200 can beused to detect drug induced-effects over time. These effects might bequalifiable/quantifiable, and may include phenomena and processes suchas changes in size parameters, necrosis, mitosis (cell splitting) etc.

The method 200 can be used for automated image analysis e.g. inhigh-throughput screening (HTS) for drug assays or the like. Themulti-parameter data set may comprise cellular data such as a microscopeimage. In various aspects of the present invention, and in contrast toconventional methods, data testing may be performed on an inter-data setbasis rather than an intra-data set basis.

The method 200 may be applied to images that have been previouslystored, transmitted etc., or may acquired and processed “on-the-fly”.The method 200 may be implemented using a processor comprising one ormore of hardware, firmware and software. For example, the processormight use a conventional personal computer (PC), ASIC, DSP, etc., and/orthe apparatus may include a GE INCELL ANALYZER 1000™ using GE's INCELLMINER™ software package upgraded to implement the method 200. Additionalfunctionality, as described herein, may also be provided by variousembodiments that implement the method 200.

In various embodiments of the present invention, the method 200 can beimplemented by an apparatus using various software components. Suchsoftware components may be provided to an apparatus in the form of anupgrade, for example, transmitted to the apparatus via the Internet.

FIG. 3 illustrates the principle of determining the Kolmogorov-Smimov(KS) distance between two data populations in accordance with variousembodiments of the present invention.

For example, the method of Altschuler et al may be used, although itdoes not itself teach the use of inter-sample KS distances for detectingcorrelation between parameters, nor use of inter-sample KS distancemeasures in combination with intra-sample distance measures for thepurposes of producing summary scores to detect phenotypic changes acrossa range of parameters.

KS distance measurements are readily generated by comparison of thefrequency distribution of data within two data sets, as seen in FIG. 3.Conversion of standard distribution histograms (data graphed as count orfrequency of data points (i.e. cells) for a given data value) tocumulative frequency distribution curves (data graphed as cumulativefraction of data for a given data value) allows two populations of datawith complex distributions to be compared as similar functions and thedifference in distributions expressed as the maximum vertical distancebetween the cumulative frequency functions; the KS distance.

Since KS distance measurement may be used without prior knowledge of thenormality or non-normality in the distribution of an analysis parameteracross a cell population, and may be used for comparing datasets ofunequal size, e.g. data from two cell populations comprising differentnumbers of cells, use of KS distance provides an un-biased comparativemeasure of differences in data derived from two cell populations, andhas been shown to significantly improve data quality from HCS assays.

Use of KS distance or other non-parametric comparators allows highlycomplex HCS datasets comprising many different parameters to be analysedand allow for example the effects of dose and/or time of exposure of acompound on cells to be accurately measured by use of the distancemeasure to determine whether data from two populations, e.g. control anddrug treated cells differ significantly.

A common problem occurring in HCS in acquisition of multi-parameter datais determining amongst the many quantitative parameters abstracted byimage analysis which are independent parameters (i.e. those parameterswhich are related to and measuring different cellular phenomena) andwhich are correlated parameters (i.e. those parameters which are relatedto and measuring the same cellular phenomena). For example inmorphological measurements of cell nuclei certain parameters might beexpected to be correlated; nuclear area, length and perimetermeasurements all relate to a single object and are relatedgeometrically. In less simple cases pairs or groups of parameters may bemeasuring the same cellular phenomena by different means not apparent tothe investigator and hence be directly or indirectly correlated. Thereis therefore a need to distinguish between measurement parameters whichare mathematically correlated and those which are biologicallycorrelated.

Undetected data correlation is a significant issue for multi-parameterassays, which are becoming standard in HCA, the increased data depthrequires multiple KS distance measures to define phenotypic differences,where a cellular phenotype may be described and defined using aphenotypic signature comprising a series of KS distance measures indifferent parameters relative to a control population. In such casesproblems may arise in interpretation of data, since a phenotypic changemay be detected in a number of parameters without knowledge ofunderlying correlation between parameters. This may lead tooverestimates of phenotypic change, or failure to discriminate betweenphenotypes, where clustering or other ranking of phenotypes is biased bycorrelated parameters (i.e. measuring the same cellular phenomena twicevia separate parameters).

Correlation of parameters does not necessarily devalue or negate the useof such parameters, provided the presence of a correlation is known. Forexample, as described above, it would be expected that parametersassociated with nuclear morphology might exhibit correlation, e.g.nuclear diameter or length would be correlated to nuclear perimeter. Ifnuclear shape were invariant, the correlation between the two parameterswould be invariant and measurement of one of the parameters would beredundant in providing information on phenotypic changes occurringwithin a cellular population. However if nuclear shape were to vary witha particular treatment the degree of correlation between the parametersis likely to change; for example, if nuclei were to change from apredominantly circular morphology to a stellate form the closecorrelation between diameter or length and perimeter would changesignificantly, the stellate nuclei having a much larger perimeter inrelation to diameter or length. Hence knowledge of the correlationsbetween parameters and detection in the change of such correlations addsto the data available to accurately characterise changes in cellularphenotypes from HCS data.

One method according to certain aspects of the present invention seeksto address the problem of parameter correlation in multi-parameter HCAdata by performing KS distance, or other applicable populationcomparator measure calculations, between samples (intra-sample) andadditionally within samples (inter-sample). The novel use ofinter-sample analysis provides means to:

-   i) establish whether two or more parameters are correlated;-   ii) use a combination of intra-sample and inter-sample distance    measures to produce phenotype maps to graphically represent    phenotypic signatures based on multi-parameter data;-   iii) use inter-sample measurements to weight and prioritise data    produced from intra-sample measurements and produce a single    phenotype score measure summarising the cumulative difference    between two cellular populations across a range of measured    parameters; and-   iv) provide additional phenotype characterisation data by    measurement of changes in parameter correlation using inter-sample    distance measures.

FIG. 4 shows an illustration of intra-sample and inter-sample KSdistance determinations as used in various embodiments of the presentinvention.

When using KS distance, or other non-parametric population comparators,it is standard practice to calculate KS distance between a test andcontrol sample, e.g. between data derived from a cell population exposedto a drug and an equivalent population of cells in the absence of thedrug. In a typical investigation this may involve exposing populationsof cells to increasing concentrations of a test substance and separatelycalculating a series of KS distances for each treated populationrelative to a control population.

Such an approach is illustrated in FIG. 4 where cell populations undertwo conditions (C₁ and C₂) are analysed for two parameters (P₁ and P₂)and the intra-sample KS distances for the two parameters are calculated(KS₁ and KS₂). Depending on the nature of phenotypic changes occurringin the samples between conditions C₁ and C₂ the intra-sample KS valuesfor the parameters will vary accordingly. In scenario A where thedistribution of parameters vary significantly between C₁ and C₂ both KS₁and KS₂ will be high. In scenario B where the distribution of P₁ variessignificantly between C₁ and C₂, but the distribution P₂ of is notsignificantly changed, KS₁ will be high and KS₂ low. Such measurementsreflect standard practice and allow quantitative measurement of thechanges in cellular parameters arising in cell population under testconditions.

A frequently encountered problem with the conventional method ofanalysis is that intra-sample KS distances (KS₁ and KS₂), whileproviding information on the differences in distributions of individualparameters between test and control samples, give no indication ofwhether two parameters show correlation and hence may be measuring thesame phenomena and so introducing redundancy into the analysis. Thiscase is illustrated in scenario A where both KS₁ and KS₂ will give highvalues, but in isolation give no indication of whether the two distancemeasures relate to the same or different cellular phenomena.

In the method of certain aspects of the present invention, the use ofintra-sample distance measures (i.e. comparison of distribution of thesame parameter under different treatment conditions), which may compriseKS distance or other suitable non-parametric distance measures, isenhanced by the use of additional inter-sample distance measures tocompare the distributions of different parameters in the same sample(i.e. comparison of distribution of different parameters under the sametreatment conditions).

These inter-sample measurements are indicated in FIG. 4 as KS₃ and KS₄and provide means for determining the degree of correlation betweenmeasurement parameters P₁ and P₂ under conditions C₁ and C₂. In scenarioA where the distribution of both parameters P₁ and P₂ change between C₁and C₂, but where both parameters have similar distributions in C₁ andC₂ suggesting a degree of correlation, measurement of inter-sample KSdistances KS₃ and KS₄ provides means for testing for such correlation.In this scenario, the relationship between intra-sample and inter-sampleKS distance values is:KS₁≈KS₂>>KS₃≈KS₄  Equation (1)wherein for intra-sample measurements the high values of KS₁ and KS₂indicate a significant change in both parameters between C₁ and C₂,while the low KS₃ and KS₄ values for inter-sample measurements indicatethat the difference between the distributions of the two parameters atboth C₁ and C₂ are similar indicating a high degree of correlationbetween P₁ and P₂.

In contrast, in scenario B only the distribution of P₁ changessignificantly between C₁ and C₂ while P₂ retains substantially the samedistribution. In this scenario, the relationship between intra-sampleand inter-sample KS distance values is:KS₁≈KS₄>>KS₂≈KS₃  Equation (2)wherein for intra-sample measurements the high values of KS₁ indicate asignificant change in the distribution of P₁ between C₁ and C₂ and thelow value of KS₂ indicates conservation of the distribution of P₂between C₁ and C₂, where for inter-sample measurements the low KS₃ valueindicates a similar distribution in P₁ and P₂ which may be indicative ofcorrelation in these parameters at C₁, and the high value for KS₄indicates the absence of correlation between P₁ and P₂ at C₂.

Consequently combination of intra-sample and inter-sample distancemeasures by a method in accordance with various aspects of the presentinvention provides new ways for:

-   i) testing for correlation between parameters (detection of low    inter-sample KS distances at all conditions); and-   ii) highlighting changes in parameter correlation between conditions    (detection of inter-sample KS distances varying across conditions).

The first aspect is important in optimising data collection in HCS;collection of correlated data (i.e. data describing the same phenomenaby different means) is a redundant activity and wastes data storage andprocessing time. The second aspect is important in maximising theinformation content of HCS data; information that two parameters arechanging correlation can yield additional information from existingparameters (e.g. variance in the correlation in object dimensionalparameters can yield information relating to changes in objectmorphology not apparent from the individual parameters).

It will be clear to those skilled in the art that further scenariosinvolving differing behaviour between two parameters are possiblewherein the two parameters exhibit varying degrees of change indistribution and correlation either under two conditions as describedabove or under a larger series of conditions where the differentconditions may represent cellular populations exposed to differentsubstances, concentrations of substances, for different times or othervarying conditions alone or in combination, such as might be encounteredin a large scale HCS screening program. Furthermore it will beappreciated by those skilled in the art that the same principlesembodied in aspects of the present invention when combining intra-sampleand inter-sample analysis may be applied to greater than two parameters,with a geometric increase in the number of pair-wise comparisonspossible for inter-sample distance calculations.

FIG. 5 shows the relationship between data parameters and phenotype mapdimensions in an embodiment of the present invention.

The application of one method according to an embodiment of theinvention to larger datasets is illustrated in FIG. 5. For any set ofHCS parameters of where the number of parameters is P, the number ofavailable pair wise inter-sample distance measurements is:

$\begin{matrix}\frac{P^{2} - P}{2} & {{Equation}\mspace{14mu}(3)}\end{matrix}$

Combining these inter-sample distance measurements for all P parameters(i.e. all distances between the distributions of each parameter at agiven treatment condition) with intra-sample distance measurements (i.e.all distances between the distributions of individual parameters at agiven treatment condition and the distribution of the same parameterunder control conditions) yields a total number of distance measurementsM:

$\begin{matrix}{M = {P + \frac{P^{2} - P}{2}}} & {{Equation}\mspace{14mu}(4)}\end{matrix}$

Where M is the number of dimensions of a graphical representation of thedata as a phenotype distance map representing all available distancemeasures for a set of parameters P and where M increases as a geometricfunction of P as illustrated in FIG. 5.

A representative phenotype distance map is shown in FIG. 5 for fourparameters P₁, P₂, P₃ and P₄. Each axis of the map is scaled from 0-1corresponding to the available range for KS distance measurements andeach axis shows the KS distance value for intra-sample measures (P₁, P₂,P₃ and P₄) and for the six available inter-sample distance measures(P₁-P₂, P₁-P₃, P₁-P₄, P₂-P₃, P₂-P₄ and P₃-P₄).

EXAMPLES

The following examples serve to illustrate embodiments of the presentinvention. These examples are intended to demonstrate techniques whichthe present inventors have found to work well in practising the presentinvention. Hence these examples are detailed so as to provide those ofordinary skill in the art with a complete disclosure and description ofthe ways in which the methods of this invention may be performed. Thefollowing Examples are intended to be exemplary only and changes,modification and alterations can be employed to the conditions describedherein, without departing from the scope of the invention.

Example 1 Generation of Multi-Parameter Cellular Data and Analysis

CHO-K1 cells were seeded into a GE Healthcare 96 well MatriPlate at 6000cells/well in Ham's F12 media supplemented with 5% EBS and incubatedunder standard tissue culture conditions for 24 hours. Mitomycin C (MMC)solutions were added in culture media to provide final concentrations inthe assay of 0-10 mM and cells incubated for a further 48 hours. Mediawas removed from wells and cells washed once with 200 ml phosphatebuffered saline (PBS), followed by fixation in ethanol for 30 minutes atroom temperature. Nuclei were stained with 5 mM HOECHST™ 33342 (Sigma)at room temperature for 15 minutes and cells washed with 200 ml PBS.

Cells were imaged by a GE Healthcare INCELL ANALYZER 1000™ using v3.5acquisition software (available from GE Healthcare) with a 20×objective. Images were acquired using on-line cell counting to acquire aminimum of 1000 cells/well. Images were analysed for nuclear area,length, perimeter and weighted moment of inertia (WMOI) using GE INCELLINVESTIGATOR™ software (available from GE Healthcare).

FIG. 6 shows representative images from the high-content screening ofcells treated with increasing concentrations of Mitomycin C.

Treatment of cells with MMC, a well known clastogenic agent causing DNAstrand breakage leads to significant changes in nuclear morphologyincluding increase in nuclear area associated with cell cycle blockagein the G2 phase of the cell cycle, formation of micronuclei throughincorrect segregation of damaged chromosomes at mitosis, and at highconcentrations, changes in nuclear shape and texture associated withnuclear fragmentation and breakdown. Changes in nuclear size, shape andmorphology are clearly visible in representative images from HCS imageacquisition shown in FIG. 6. Images A, B and C show typical nuclearmorphology for cells treated with 0, 0.16 and 2.5 μM MMC respectively,and the inset image in image B (0.16 μM MMC) shows a highermagnification view of nuclei with associated micronuclei arising fromclastogenic DNA damage.

Four nuclear morphology parameters were abstracted from images usingautomated image analysis and data from replicate samples exposed to thesame concentrations of MMC combined to yield population distributionhistograms for each of the four parameters as shown in FIGS. 7 to 10. Ineach of FIGS. 7 to 10 the data in panel 1 is from control cells, and thedata in panels 2-12 from MMC treated cells across the range of 0.01μM-10 μM MMC.

KS distance analysis of the data using intra-sample measurements (i.e.calculation of the KS distance between cell data in the absence of MMCand cell data at a given concentration of MMC across the range 0.01μM-10 μM) was performed and the resulting data shown in FIG. 11. Thedata shows a clear dose-dependent increase in KS distances for all fourmeasured parameters indicating a significant dose-dependent change inthese parameters relative to untreated cells. Three parameters, nucleararea, length and perimeter showed almost identical KS distances fromuntreated cells across the whole MMC concentration range. WMOI (ameasure of the distribution of the DNA staining pixel intensity valueswithin the nucleus) also showed dose-dependent changes, but to adiffering degree to the other three measured parameters.

Inter-sample KS distances were calculated for all six possible parameterpairings (Area-Length; Area-WMOI; Length-WMOI; Area-Perimeter;Length-Perimeter; Perimeter-WMOI) by calculation of KS distance betweendata distributions for two parameters at the same MMC concentration. Inorder to perform KS distance measures on the different parameters ofwhich had variant dimensions, parameter values were normalized(P_(norm)) to a range from 0-1 using the function:

$\begin{matrix}{P_{norm} = \frac{P - P_{\min}}{P_{\max} - P_{\min}}} & {{Equation}\mspace{14mu}(5)}\end{matrix}$

Results for inter-sample KS measurements are shown in FIG. 12 (pair wisedistance measures are indicated as follows; A-L Area-Length; A-WArea-WMOI; L-W Length-WMOI; A-P Area-Perimeter; L-P Length-Perimeter;P-W Perimeter-WMOI). The data shows a number of clear patterns in therelationship between parameters revealed by inter-sample KS distancecalculations including but not restricted to:

-   i) Area and length (A-L) show a constant low KS distance across the    entire MMC concentration range indicating that these two parameters    show similar distributions with a high degree of correlation. Since    these two morphological measures are both directly influenced by    nuclear size this data indicates that while nuclei are increasing in    size on MMC treatment, they are not undergoing morphological changes    that change the relationship between area and length (e.g. changing    from circular to ovoid);-   ii) Length and WMOI (L-W) show a constant high KS distance over most    of the MMC concentration range indicating that these two parameters    have a low degree of correlation, i.e. DNA intensity distribution    within nuclei is not related to nuclear length;-   iii) Perimeter and WMOI (P-W) show a dose dependent change in KS    distance in an inverse manner to that observed for intra-sample KS    distances for these parameters, i.e. the inter-sample KS distance    between the distributions of perimeter and WMOI decreases with MMC    concentration indicating that these two parameters while not    correlated at low dose become correlated at high dose, implying a    change in phenotype involving some form of linkage between    increasing nuclear perimeter and DNA distribution; and-   iv) Area and perimeter (A-W) show a low KS inter-sample distance at    low and high MMC concentrations with an increase in KS distance at    intermediate concentrations, indicating the possibility of two or    three phenotype classes occurring at low, medium and high MMC    concentrations.

It is well-known that treatment of cells with agents such as MMC thatinduce DNA damage leads to a complex series of events occurring withincells that results in a range of phenotypes which are related to MMCconcentration. At low concentrations cells can repair DNA damage withlittle or no effect on cell cycle progression and minor variations innuclear morphology and micronuclei formation. At higher concentrationsof MMC the consequently higher levels of DNA damage impose a higher DNArepair burden on cells which results in significant cell cycle delaysand an increase in micronuclei formation and other alterations innuclear structure. At still higher doses of MMC the extent of DNA damagebecomes sufficiently high to trigger cells to undergo apoptosisaccompanied by gross changes in nuclear morphology and nuclearbreakdown.

All of these interrelated processes lead to a very complex series ofevents occurring in MMC treated cells which impinge directly orindirectly on the observed phenotypes at different concentrations ofMMC. The complexity of the events taking place within a large populationof cells is not fully apparent from traditional analytical approachessuch as analysis of intra-sample population data distribution (FIG. 11).Application of a method in accordance with aspects of the presentinvention using additional inter-sample population data distributioncomparators (FIG. 12) reveals patterns in the data indicative ofphenotypic changes which are not apparent in conventional analyses.

Combination of intra-sample and inter-sample distance measures may beused to generate phenotype maps (FIG. 13) summarizing the variations andinterrelationships in measured parameters at different treatmentconditions. Such maps allow rapid visual assimilation of the trends andinterrelationships in HCS analysis data not fully apparent fromconventional graphical representations such as dose-response plots.

As described previously, and illustrated in FIG. 5, the complexity ofphenotype distance maps grows geometrically with the number of analysedparameters, however these complex maps may be broken down into a seriesof simpler elements to aid in further analysis as shown in FIG. 14.

In the simplest case (FIG. 14A) a phenotype map derived from threeparameters has three axes, one for the intra-sample distance measure foreach of the parameters (P₁ and P₂) and one for the only availablepair-wise inter-sample distance measure (P₁-P₂). Increasing the numberof analysed parameters to three increases the complexity of the map to asmall degree (FIG. 14B) yielding six axes, one for the intra-sampledistance measure for each of the parameters (P₁, P₂ and P₃) and threefor the pair-wise inter-sample distance measures (P₁-P₂, P₁-P₃ andP₂-P₃). Further increasing the number of analysed parameters increasesthe number of distance map axes as described previously where the numberof axes M is related to the number of parameters P by Equation 4.

Whatever the number of parameters and the consequent level of complexityof the phenotype distance map, any such map be broken down into a numberof simple components each representing a single pair wise comparison.Hence as shown in FIG. 14 a map derived from analysis of threeparameters where M=6 (FIG. 14B) may be broken down in to a series ofconstituent elements B₁, B₂ and B₃. For any given phenotype map ofdimension M the number of constituent elements is as defined by Equation3.

Breaking down the phenotype map into components based on pair wisecomparisons allows individual pairings to be given a phenotype changescore (S) based on the intra-sample (D) and inter-sample distance (d)scores for the parameter pair:S=d(D _(P1) +D _(P2))  Equation (6)wherein the score is subject to a weighting based on the inter-sampledistance calculated for the pairing, i.e. two parameters with a highinter-sample distance indicating a lack of correlation will produce ahigher score than the same parameter values accompanied by a lowinter-sample distance score.

For complex maps comprising greater that one component these scores canbe calculated for each component and an overall phenotype change scorecalculated by summing the individual scores to yield a cumulative scoretaking account of all parameters measured in a HCS analysis. Cumulativephenotype scores for cells treated with a range of MMC concentrationsand analysed for four nuclear morphology parameters are shown in FIG.15.

It will be readily understood by those skilled in the art that furthertypes of data comparison and cumulative scoring are possible based onthe principles of combining intra-sample and inter-sample KS distancemeasures as described in the method according to aspects of the presentinvention. For example, the additional inclusion of base lineinter-sample distance measures whereby KS distance measures obtainedbetween different parameters in a control population in order toestablish the degree of correlation between parameters allows furtherpair-wise comparisons and phenotype scoring to be undertaken.

Using the additional factor of control population inter-sample distancemeasures allows individual pairs of parameters to be given a phenotypechange score (S) based on the intra-sample (D) distance scores for bothparameters combined with inter-sample distance scores for both control(d_(c)) and treated (d_(t)) populations where the score takes intoaccount the degree of correlation in the parameters under the twoconditions:S=(d _(c) +d _(t))×(D _(P1) +D _(P2))  Equation (7)

This approach allows correlation changes across a range of conditions tobe taken into account. For example, in a scenario where two parametersare not correlated in either control or test samples (d_(c) and d_(t)are both high) the resulting score will be maximised, conversely if thetwo parameters are correlated in both control and test samples (d_(c)and d_(t) are both low) the resulting score will be minimised.

Performing this operation for all parameter pairs and summing to producea cumulative score provides a way to summarise data based on all HCSparameters measured.

The forgoing methods and other related embodiments of the presentinvention which encompass the use of both inter-sample and intra-samplepopulation distribution distance measures allow very complex HCSdatasets to be consolidated and summarised to measures which can bereadily compared across large scale screening programs. The methods ofvarious aspects and embodiments of the present invention are useful forevaluation of large scale drug, RNAi or other screening programs wherethe consolidation of multi-parameter data by such methods allowscreening hits to be identified on the basis of combined phenotypicparameters and differential effects of treatment producing differentphenotype classes, identified by different cumulative phenotype scoresto be segregated.

By removing correlated parameters from a final analysis parameter set,various aspects and embodiments of the present invention can, forexample, improve analysis efficiency by running small scale analysis,determining correlated parameters and then dropping the correlatedparameters from an analysis parameter set that is subsequently used fora larger scale analysis.

Various aspects and embodiments of the present invention also, oralternatively, allow for modifying parameter values with respect tomeasured correlation, whilst retaining the modified parameters so thatphenotypic scores comprise weighted aggregates of descriptive parameterssuch that correlated parameters do not overly influence the phenotypicscore(s).

Whilst the present invention has been described in connection withvarious embodiments, those skilled in the art will be aware that manydifferent embodiments and variations are possible. All such variationsand embodiments are intended to fall within the scope of the presentinvention as defined by the appended claims.

What is claimed is:
 1. A method for automated high-content screening(HCS) of a plurality of samples comprising cells and subjected to sampletreatment conditions in an assay, wherein the plurality of samplescomprise both samples subjected to a same treatment condition andsamples subjected to different sample treatment conditions, the methodcomprising: processing data from each of two or more images of theplurality of samples to obtain a multi-parameter data set for eachimage; for samples subjected to the same sample treatment condition,obtaining inter-sample measurements based on distributions of differentparameters in the same sample under the same sample treatment condition;for samples subjected to different sample treatment conditions,obtaining intra-sample measurements based on distributions of sameparameter under the different sample treatment conditions; determining,based on both inter-sample and intra-sample measurements, correlatedparameters in the respective multi-parameter data sets, whereinparameters from the multi-parameter data sets are graphically visualizedin a phenotype map generated based on the inter-sample and intra-samplemeasurements and compared to determine the correlated parameters, andwherein correlation is to be determined using a Kolmogorov-Smirnov (KS)distance measurement analysis of parameters from the multi-parameterdata sets; creating an analysis parameter set by removing at least oneof the correlated parameters from the multi-parameter data sets to formthe analysis parameter set; applying the analysis parameter set toidentify one or more cellular phenotypes of the samples from themulti-parameter data sets; and providing an identification, in theplurality of samples, of one or more cells that undergo changes incellular phenotypes.
 2. The method of claim 1, further comprisingidentifying one or more phenotypes from a plurality of multi-parameterdata sets and comparing respective phenotypes from the multi-parameterdata sets to identify variations therebetween.
 3. The method of claim 1,further comprising forming one or more respective analysis parametersets for a plurality of multi-parameter data sets, and comparing theanalysis parameter sets to determine whether or not a correlationrelationship is maintained between the multi-parameter data sets.
 4. Themethod of claim 1, wherein correlation is determined by the KS distancemeasurement analysis made between the phenotypes in a predeterminedmulti-parameter data analysis set.
 5. The method of claim 4, wherein theKS distance measurement analysis includes a comparison of the frequencydistribution of data within two data sets in the predeterminedmulti-parameter data analysis set.
 6. The method of claim 1, whereinmodifying correlated parameter values within the predeterminedmulti-parameter data analysis set comprises removing one of a pair ofparameters whose correlation exceeds a predetermined threshold valuefrom the analysis parameter set.
 7. A tangible non-transient computerprogram storage product comprising machine instructions operable toconfigure a data processing apparatus to: process data from each of twoor more images of a plurality of samples to obtain a multi-parameterdata set for each image; for samples subjected to a same sampletreatment condition, obtain inter-sample measurements based ondistributions of different parameters in the same sample under the samesample treatment condition; for samples subjected to different sampletreatment conditions, obtain intra-sample measurements based ondistributions of same parameter under the different sample treatmentconditions; determine, based on both inter-sample and intra-samplemeasurements, correlated parameters in the respective multi-parameterdata sets, wherein parameters from the multi-parameter data sets aregraphically visualized in a phenotype map generated based on theinter-sample and intra-sample measurements and compared to determine thecorrelated parameters, and wherein correlation is to be determined usinga Kolmogorov-Smirnov (KS) distance measurement analysis of parametersfrom the multi-parameter data sets; create an analysis parameter set byremoving at least one of the correlated parameters from themulti-parameter data sets to form the analysis parameter set; apply theanalysis parameter set to identify one or more cellular phenotypes ofthe samples from the multi-parameter data sets; and provide anidentification, in the plurality of samples, of one or more cells thatundergo changes in cellular phenotypes.
 8. A system for automatedhigh-content screening (HCS) of a plurality of samples comprising cellsand subjected to sample treatment conditions in an assay, wherein theplurality of samples comprise both samples subjected to a same treatmentcondition and samples subjected to different sample treatmentconditions, the system comprising: an imager for obtaining two or moreimages of the plurality of samples, each image represented by arespective multi-parameter data set; and an image processor that isoperable to: process data from each of the two or more images to obtainrespective ones of the multi-parameter data sets; for samples subjectedto the same sample treatment condition, obtain inter-sample measurementsbased on distributions of different parameters in the same sample underthe same sample treatment condition; for samples subjected to differentsample treatment conditions, obtain intra-sample measurements based ondistributions of same parameter under the different sample treatmentconditions; determine, based on both inter-sample and intra-samplemeasurements, correlated parameters in the respective multi-parameterdata sets, wherein parameters from the multi-parameter data sets aregraphically visualized in a phenotype map generated based on theinter-sample and intra-sample measurements and compared to determine thecorrelated parameters, and wherein correlation is to be determined usinga Kolmogorov-Smirnov (KS) distance measurement analysis of parametersfrom the multi-parameter data sets; create an analysis parameter set byremoving at least one of the correlated parameters from themulti-parameter data sets to form the analysis parameter set; apply theanalysis parameter set to identify one or more cellular phenotypes ofthe samples from the multi-parameter data sets; and provide anidentification, in the plurality of samples, of one or more cells thatundergo changes in cellular phenotypes.
 9. The system of claim 8,wherein the image processor is further operable to identify one or morephenotypes from a plurality of multi-parameter data sets and comparerespective phenotypes from the multi-parameter data sets to identifyvariations therebetween.
 10. The system of claim 8, wherein the imageprocessor is further operable to form two or more respective analysisparameter sets for a plurality of multi-parameter data sets, and comparethe analysis parameter sets to determine whether or not a correlationrelationship is maintained between the multi-parameter data sets. 11.The system of claim 8, wherein the image processor is further operableto determine correlation by using the KS distance measurement analysismade between the parameters in a predetermined multi-parameter dataanalysis set.
 12. The system of claim 11, wherein the KS distancemeasurement analysis includes a comparison of the frequency distributionof data within two data sets in the predetermined multi-parameter dataanalysis set.
 13. The system of claim 8, wherein the image processor isfurther operable to modify correlated parameter values within apredetermined multi-parameter data analysis set by removing one of apair of parameters whose correlation exceeds a predetermined thresholdvalue from the analysis parameter set.
 14. The system of claim 8,wherein the two or more images comprise cellular images and the imagercomprises a cellular imager.
 15. The system of claim 14, wherein eachmulti-parameter data set includes one or more parameters of: nucleararea; length; diameter; perimeter; ellipticity; and weighted moment ofinertia (WMOI), form, and distribution.
 16. The system of claim 14,wherein the intra-sample measurements are used in weighting andprioritizing data produced from the inter-sample measurements to producea single phenotype score measure summarizing a cumulative differencebetween two cellular populations across a range of measured parameters.17. The system of claim 8, wherein the image processor is furtherconfigured to produce one or more phenotype maps graphicallyrepresenting phenotypic signatures based on intra-sample measurementsand inter-sample measurements.
 18. The system of claim 17, wherein theinter-sample measurements and the intra-sample measurements comprisepopulation comparator measures.
 19. The system of claim 18, wherein thepopulation comparator measures comprise Kolmogorov-Smirnov (KS) distancemeasurements.
 20. The system of claim 8, wherein the intra-samplemeasurements are utilized to measure changes in the inter-samplemeasurements to provide phenotype characterization data.
 21. The systemof claim 8, wherein the sample is subjected to a drug treatment.
 22. Thesystem of claim 8, wherein the sample comprises a cellular populationand the two or more images comprises a microscope image of the cellularpopulation.
 23. The system of claim 8, wherein identifying cells thatundergo changes in cellular phenotypes comprises detecting changes ininterrelationships of correlated parameters based on inter samplemeasurement.
 24. The system of claim 8, the image processor is furtheroperable to generate, based on a combination of inter-sample andintra-sample measurements, phenotype maps graphically representingphenotypic signatures.